1. Field of the Invention
This invention affords nucleic acid (DNA/RNA) sequencing gel compositions and a method for using same. The composition comprises an aqueous gel of one or more polysaccharides, one or more denaturing agents, one or more optional non-gelling additives, and buffers.
2. Description of Related Art
Techniques for sequencing DNA (deoxyribonucleic acid) were discovered in the late 1970's and have become very important tools in molecular biology. The basic technology for sequencing DNA, and newer methods which may involve other forms of nucleic acid such as RNA (ribonucleic acid), may be divided into two steps for the purpose of this invention. First, a set of single-stranded nucleic fragments is generated. Current techniques in wide use for this step include the well-known methods of Sanger and of Maxam & Gilbert, as well as newer methods such as "cycle sequencing". Second, the fragments produced by any of these techniques are separated by molecular weight by gel electrophoresis. The resulting pattern of "bands" of DNA fragments separated by size is then "read" (interpreted) to determine the nucleotide sequence of the original DNA in the reaction. The original gel medium for performing this separation was the crosslinked polyacrylamide gel, containing high levels of urea to minimize formation of secondary structure in the DNA fragments. There has been very little change in the formulation and method of manufacture of the polyacrylamide gel since the beginning of the technique. Traditional sequencing gels are tedious to make. It would clearly be of use to laboratory researchers to have these complex gels prefabricated, to reduce the labor and uncertainty of making them. However, the traditional gel has proven to have poor storage stability, and cannot in general be kept over about a week, even at 4.degree. C., before suffering loss of resolution. The stabilization of polyacrylamide gels is difficult and tedious, as discussed in U.S. Pat. No. 5,159,049--Allen. Attempts have been made to use other polymers for the sequencing gel--for example, to use substituted acrylamides in place of acrylamide, or to use improved crosslinkers [see U.S. Pat. No. 5,055,517--Schorr, et al.; and EPA 89/115 833.9]; and U.S. Pat. No. 5,073,603--Ponticello discloses oxygen-tolerant methods for making acrylamide gels. However, none of these is known to be available as a storage-stable sequencing gel.
It is essential in DNA sequencing to use a gel which can separate bands having a molecular weight difference of only one nucleotide. This can be an extremely demanding separation. To separate DNA fragments with 100 as compared to 101 bases requires a resolution value of 1%; a more typical requirement is separation of 200 and 201 bases giving a resolution value of 0.5 %; separations in the range 300 to 400 bases giving a resolution value as low as 0.25% are at the limit of present technology with a single loading.
It has been known for decades that agarose forms a very stable gel. Agarose gels can be kept for years with little change in properties, as long as the moisture level is maintained. However, agarose has been considered unsuitable as the primary gel polymer in sequencing gels. One school of thought has believed that agarose is too poorly sieving to give adequate separation in the very demanding DNA sequencing application [see U.S. Pat. No. 4,857,163--Gurske, et al., where acrylamide is "too sieving" for an agarose use; U.S. Pat. No. 4,319,975--Cook, distinguishing uses for agarose as compared to acrylamide; U.S. Pat. No. 3,766,047--Elevitch, noting that agarose can be too difficult to use for "fine resolution" of one protein from another (which is much less demanding than DNA sequencing), and U.S. Pat. No. 5, 159,049--Allen, at col. 1 line 66. Another common perception is that agarose does not form gels in strong urea solutions [see U.S. Pat. No. 4,774,093--Provonchee, et al.; Hoffman, et al., "Electrophoresis" 10:741-747 (1989); " SeaNotes" (FMC Corporation, Winter 1980-81 )]. This is not rigorously correct, because Smith et al., in "Nucleic Acids Research" 9:5269 - 5286, (1981) made solutions of 1.75% agarose in 7 M urea and acid citrate buffer (0.025 M citric acid; pH 4.5), chilled them at 4.degree. C. to make them set into gels, and used the gels to separate large RNA molecules by electrophoresis. The gels were run at 4.degree. C. In addition, the low current level (25 mA) at low ionic strength, and consequently long running time (30 hrs.), prevented heat generation during electrophoresis. Of especial interest is that the 7 M urea did not totally remove secondary structure in the RNA; when secondary structure was completely abolished by use of aldehydes, then the RNA separation failed. Also, Locker ["Analyt. Blochem." 98:358-367, (1979)] separated RNA on agarose gels in the presence of 6 M urea and in neutral solution. The gels were cooled to set them, and run at low voltage (100V for a 20 cm gel, compared to 1000 V for a sequencing gel) and hence at a lower temperature than required for sequencing. Locker notes (p. 364) that the RNA secondary structure is not completely removed in these gels, and that they are therefore not useful for determining molecular weight, even though useful for separating different classes of RNA differing widely in molecular weight, such as transfer compared to messenger and cytoplasmic compared to mitochondrial. These uses are similar to the traditional use of agarose gels for separation of double-stranded DNA fragments of moderate size (about 100 to 10,000 base pairs.)
In contrast, in the sequencing of DNA it is essential to abolish all secondary structure in the DNA being analyzed, so that separation is strictly on the basis of molecular size, since otherwise the sequence cannot be read accurately [see R Frank, et al., "Nucleic Acid Research" 9:4967 -4979, (1981)]. In traditional acrylamide gels, complete denaturation of DNA, to optimize resolution, is accomplished by two mechanisms: (1) by running sequencing gels at elevated temperatures; and (2) by incorporation of a high level of urea, which is partially effective in abolishing secondary structure. It may be noted however, that even in acrylamide, "compression zone" regions of DNA with multiple base repeats do not resolve well. Resolution of compressed zones is normally accomplished by using high levels of current and voltage, so that the gels are kept warm by Joule heating. Typical voltages are 1000 -2000V, (with interelectrode spacing of about 50 cm) or over 10 times the voltages normally used to separate RNAs or double-stranded DNA fragments. Acrylamide has been satisfactory for this use because its coherence as a gel is via covalent crosslinks that are not dissolved by heat or denaturants. However, as noted above, acrylamide has serious problems of chemical stability that prevent prolonged storage of pre-cast gels. Other gelling materials have been used to separate nucleic acids [see for example, JP 4-248460], but separation of double-stranded restriction fragments (differing by tens of base pairs) is shown, rather than the single-base resolution needed for sequencing.
It also may be noted that a prominent text in the field ["Molecular Cloning"--2nd ed, by Sambrook, Fritsch & Maniatis, Cold Spring Harbor Laboratory Press, (1989)] in its discussion of Gel Electrophoresis of DNA--Chapter 6, states at 6:36-37: "Polyacrylamide gels are more of a nuisance to prepare and run than agarose gels. ... However, they have three major advantages over agarose gels: (1) Their resolving power is so great that they can separate molecules of DNA whose lengths differ by as little as 0.2% (i.e., 1 bp in 500 bp)...".